Microbial production of pyruvate and pyruvate derivatives

ABSTRACT

Microbial production of pyruvate and metabolites derived from pyruvate in cells exhibiting reduced pyruvate dehydrogenase activity compared to wild-type cells. Acetate and glucose are supplied as a carbon sources.

This application is a continuation-in-part of International ApplicationNo. PCT/US03/05083, filed Feb. 20, 2003, which claims the benefit ofU.S. Provisional Application No. 60/359,279 (filed Feb. 20, 2002) andU.S. Provisional Application No. 60/402,747 (filed Aug. 12, 2002), allof which are incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

In stating a vision for the future in “Biobased Industrial Products,Priorities for Research and Commercialization”, the National ResearchCouncil has proposed U.S. leadership for the global transition tobiobased products. The report provides compelling evidence for acompetitively priced biobased products industry that will eventuallyreplace much of the petrochemical industry. In light of this report andthe desirability of reducing U.S. reliance on foreign oil, there is anincreasing interest in generating commodity and fine chemicals fromwidely available U.S. renewable resources, e.g., crops, throughfermentation. In the last few years, companies have invested hundreds ofmillions of dollars in commercializing the microbial production ofseveral biochemicals, such as lactic acid.

Microbial fermentation processes are used to generate a wide variety ofimportant biochemicals such as ethanol and lysine (markets in thebillions of U.S. dollars). In order to be economic, fermentations relyon microorganisms which have been developed by selection or geneticmeans to accumulate a specific product that is produced via metabolism.Microbial metabolic pathways are not naturally optimal for thegeneration of a desired chemical, but have instead evolved for thebenefit of the organism. Metabolic engineering is the targeted andrational alteration of metabolism, and it involves the redirection ofcellular activities to generate a new product or generate a product at ahigher rate or yield.

Pyruvate (pyruvic acid) is a three-carbon ketoacid synthesized at theend of glycolysis. Pyruvate is an important raw material for theproduction of L-tryptophan, L-tyrosine, 3,4-dihydroxyphenyl-L-alanine,and for the synthesis of many drugs and biochemicals. Pyruvate has usein the chemical industry and finds wide application in cosmetics.Clinical studies have found that pyruvate can promote weight loss andfat loss, hence it is commonly marketed in tablet form as a dietarysupplement. Recent research indicates that pyruvate also functions as anantioxidant, inhibiting the production of harmful free radicals.

Certain microorganisms have been found to produce useful quantities ofpyruvate from glucose, an inexpensive substrate derived from cornstarch. The yeasts Debaryomyces coudertii (M. Moriguchi, Agr. Biol.Chem. 46: 955-961 (1982)) and Saccharomyces exiguus (A. Yokota et al.,Agr. Biol. Chem. 48: 2663-2668 (1984)), for example, are known toaccumulate pyruvate, as are the basidiomycetes Schizophyllum commune (S.Takao et al., J. Ferm. Tech. 60: 277-280 (1982)), and Agricus campestris(A. Yokota et al., Agr. Biol. Chem. 48: 2663-2668 (1984)). The yeaststrain Torulopsis glabrata IFO 0005 was found to be a superior strainfor the production of pyruvate (T. Yonehara et al., J. Ferm. Bioeng. 78:155-159 (1994)), accumulating 67.8 g/L pyruvate in 63 hours (yield0.494) in a fed-batch fermentation with successive additions of glucose(R. Miyata et al., J. Ferm. Bioeng. 82: 475-479 (1996)). A higher yield(0.673) of pyruvate was observed in T. glabrata ACII-33, a mutant withdecreased pyruvate decarboxylase (PDC) activity (R. Miyata et al., J.Biosci. Bioeng. 88: 173-177 (1999)). Decreased PDC activity preventedthe formation of acetate via acetaldehyde and thus increased thepyruvate production. T. glabrata ACII-33 accumulated 60.3 g/L pyruvatein 47 hours in a 3 L jar fermenter.

Bacteria of the genera Corynebacterium (A. Yokota et al., Agr. Biol.Chem. 48: 2663-2668 (1984)) and Acinetobacter (Y. Izumi et al., Agr.Biol. Chem. 46: 2673-2679 (1982)), Enterobacter aerogenes (A. Yokota etal., Agr. Biol. Chem. 48: 705-711 (1989)), and Escherichia coli (A.Yokota et al., Appl. Microbiol. Biotech. 41: 638-643 (1994)) are alsoknown to accumulate pyruvate. A lipoic acid auxotroph of E. coli (strainW1485lip2) was found to produce pyruvate aerobically from glucose underlipoic acid deficient conditions. This strain accumulated 25.5 g/Lpyruvate in 32 hours with a yield of 0.51 in polypepton (4 g/L)supplemented media (A. Yokota et al., Appl. Microbiol. Biotech. 41:638-643 (1994)).

Alanine, which is derived from pyruvate, is an alpha amino acid that isalso commercially important, for example as a starting material in thechemical industry. L-alanine is a chiral building block being one of thesmallest chiral compounds, with four important functional groups:hydrogen, methyl, amino, and carboxylic acid. Presently, alanine isproduced using metabolically engineered Corynebacterium orBrevibacterium bacterial strains in which alanine dehydrogenase isoverexpressed. However, the efficiency of this method of production islimited because large quantities of carbon move from pyruvate toacetyl-CoA and therefore are unavailable for alanine production.

Diacetyl, also derived from pyruvate, is a flavoring/texture agent fordairy products, and could find additional use in food products.

These compounds (pyruvate, alanine and diacetyl) have current marketprices from $10 to $50/pound. Improved production methods from renewableresources would open new markets for pyruvate and its derivatives andthus reduce reliance on petroleum-derived products.

SUMMARY OF THE INVENTION

The present invention is directed to a method for efficient microbialproduction of pyruvate and its derivatives, such as alanine anddiacetyl. The method utilizes bacterial cells exhibiting reducedactivity of at least one enzyme in the pyruvate dehydrogenase (PDH)complex of enzymes, compared to wild-type bacterial cells. The bacterialcells are cultured in the presence of a primary carbon source,preferably glucose, and a secondary carbon source such as acetate and/orethanol. If desired, the bacterial cells can be cultured in the presenceof an additional carbon source, preferably a compound that is part ofthe tricarboxylic acid cycle of the bacterial cell, such as succinate.

Preferably PDH activity in the bacterial cells is undetectable. In aparticularly preferred embodiment of the invention, the method utilizesbacterial cells wherein the gene encoding at least one enzyme in the PDHcomplex of enzymes is knocked out.

When the method of the invention is employed to produce pyruvate,pyruvate is preferably produced in an amount of at least about 30 g/L,and the yield of pyruvate as a function of glucose consumed ispreferably at least about 0.70.

Optionally the bacterial cells utilized in the method of the inventionexhibit reduced phosphoenolpyruvate carboxylase (PEP carboxylase)activity or reduced pyruvate oxidase activity, or both, compared towild-type levels.

The production of pyruvate and diacetyl according to the invention isnot redox balanced and thus NADH will accumulate in the cells. In theseembodiments, the bacterial cells can be further engineered exhibitincreased or added NADH oxidase activity compared to wild-type levels inorder to maintain redox balance. When the method of the invention isemployed to produce diacetyl, the bacterial cells also preferablyexhibit added or increased acetolactate synthase activity compared towild-type cells.

The production of alanine according to the invention is redox balancedand NADH will not usually accumulate in the cells. Production of alaninecan, however, be enhanced by utilizing bacterial cells that exhibitadded or increased alanine dehydrogenase activity and/or reduced lactatedehydrogenase activity compared to wild-type cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the biochemical pathways involved in the accumulationof pyruvate, alanine and diacetyl. Not all biochemical reactions orcofactors are shown.

FIG. 2 shows graphs of cell growth, metabolite production and feedconsumption as a function of time in fermentations using media C forstrains (a) CGSC4823; (b) CGSC4823 Δppc; (c) CGSC6162; and (d) CGSC6162Δppc.

FIG. 3 shows graphs of cell growth, metabolite production and feedconsumption as a function of time in fermentations using CGSC6162 at (a)pH of 6.0 and (b) pH of 7.0.

FIG. 4 shows a graph of fermentation as a function of time using CGSC6162 at 42° C. and at a pH of 7.0.

FIG. 5 shows a graph of fermentation as a function of time using CGSC6162 Δppc at 32° C. and at a pH of 7.0.

FIG. 6 shows a graph of cell growth, alanine production and glucoseconsumption as a function of time in a fermentation using CGSC6162modified to overexpress the alaD gene from Bacillus sphaericus.

FIG. 7 shows a graph of cell growth, alanine production and glucoseconsumption as a function of time in a fermentation using an ldhAdeletion mutant of CGSC6162 modified to overexpress the alaD gene fromB. sphaericus.

FIG. 8 shows a graph of the production of alanine in CGSC6162 ldhApTrc99A-alaD using modified growth parameters and supplemented withadditional NH₄Cl: (a) glucose (O), alanine (▪), pyruvate (□); (b) OD(●), succinate (Δ), acetate (∇).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a process that utilizes bacterial cells for theproduction of the metabolite pyruvate. The cells are grown, preferablyaerobically, in the presence of a primary carbon source such as thecarbohydrate glucose, a secondary carbon source such as acetate orethanol, and optionally a carbon source in the tricarboxylic acid (TCA)cycle such as succinate.

The process can also be used to produce metabolites derived frompyruvate (i.e., pyruvate derivatives), such as alanine and diacetyl.Metabolites “derived from” pyruvate or “pyruvate derivatives” are thosebiochemicals with respect to which pyruvate is a metabolic precursor inbacterial metabolism. In other words, the metabolic pathways for theproduction of biochemicals “derived from” pyruvate branch away from theglycolytic/TCA pathway at pyruvate. Examples of other products derivedfrom pyruvate thus include 2,3 butanediol, acetoin, isoleucine andvaline. The general microbial pathways for the synthesis of pyruvate andproducts derived from pyruvate are shown in FIG. 1.

The PDH complex includes pyruvate dehydrogenase, dihydrolipoamideacetyltransferase and dihydrolipoamide dehydrogenase. In E. coli, apreferred microbe for use in the invention, these enzymes are encoded bythe aceE, aceF and lpd genes, respectively. The bacterial cells used inthe method of the invention exhibit reduced activity of at least oneenzyme in the pyruvate dehydrogenase (PDH) complex of enzymes comparedto wild-type cells. This is referred to herein as reduced “PDHactivity.”

“Reduction” in an enzymatic activity compared to wild-type levels ofthat activity in a cell includes, but is not limited to, completeelimination of enzymatic activity. Thus, a reduction in PDH activitycompared to wild-type levels of PDH activity includes, but is notlimited to, complete elimination of PDH activity. Complete “elimination”of PDH activity encompasses a reduction of PDH activity to such aninsignificant level that essentially no carbon flows from pyruvate toacetyl CoA. Preferably, the bacterial cells used in the method of theinvention exhibit no detectable activity of at least one enzyme in thePDH complex during the entire period of fermentation. It should beunderstood that although the method of the invention is not limited bythe way in which or the extent to which PDH activity is reduced in thebacterial cells, it is preferred that PDH activity be completelyeliminated by disrupting the function of one or more genes associatedwith PDH activity. In a preferred method of the invention, bacterialcell growth surprisingly continues, with the concomitant production ofpyruvate and its derivatives, even though PDH activity is completelyeliminated.

Methods for reducing or eliminating PDH activity include those that actdirectly on the gene encoding one or more of the PDH enzymes, the mRNAtranscript produced by the gene, the translation of the mRNA transcriptinto the protein, or the abolishment of the activity of the translatedprotein. One way the activity of an enzyme can be reduced is byphysically altering the gene encoding the enzyme. For example, a geneencoding the enzyme can be mutated using site-directed mutagenesis tointroduce insertions, deletions and/or substitutions. Alternatively,transcription of a gene can be impeded by delivering to the cell anantisense DNA or RNA molecule or a double stranded RNA molecule. Anotherway the activity of an enzyme can be reduced is by interfering with themRNA transcription product of the gene. For example, a ribozyme (or aDNA vector operably encoding a ribozyme) can be delivered to the cell tocleave the target mRNA. Antisense nucleic acids and double stranded RNAsmay also be used to interfere with translation. Antibodies orantibody-like molecules such as peptide aptamers can be used to abolishthe activity of the translated protein. In general, methods that preventproduction of an active PDH enzyme yield bacterial cells that arereferred to as “gene knockouts” as the term is used herein.

Phosphoenolpyruvate carboxylase (PEP carboxylase) convertsphosphoenolpyruvate (PEP), the metabolic precursor to pyruvate, tooxaloacetate and is encoded by the ppc gene. Accordingly, the bacterialcells used in the method of the invention are optionally furthermodified to reduce or eliminate the activity of PEP carboxylase. Theinvention is not intended to be limited by the method selected to reduceor eliminate PEP carboxylase activity.

Pyruvate oxidase converts pyruvate into acetate and is encoded by thepoxB gene. Alternatively or in addition, the bacterial cells are furthermodified to reduce or eliminate the activity of pyruvate oxidase.

Hence, the method of the invention preferably utilizes bacterial cellsexhibiting, compared to wild-type cells, reduced or no PDH activity(pdh⁻) and, optionally, one or more of reduced or no pyruvate oxidaseactivity (pox⁻), reduced or no PEP carboxylase activity (ppc⁻).

In the presence of only glucose as the carbon source, bacterial cellsdeprived of PDH activity (so as to prevent conversion of pyruvate toacetyl CoA) and, optionally, PEP carboxylase (so as to prevent theprecursor of pyruvate from being depleted) and, optionally, pyruvateoxidase (so as to prevent conversion of pyruvate to acetate) wouldcertainly not be expected to grow, as these modifications adverselyaffect the ability of the cells to produce biochemical intermediatesnecessary for cell growth. In particular, complete removal of PDHactivity would prevent cells from growing on glucose due to an inabilityto generate acetyl CoA, α-ketoglutarate and succinate, all necessary forcell growth.

The fact that the cells did grow when acetate was added as aco-substrate was surprising as those skilled in the art of bacterialfermentations view glucose as a preferred carbon source over acetate.O'Beirne et al., Bioprocess Eng. 23:375-380 (2000), discuss in detailthe impact of acetate as a co-substrate in continuous cultivations of E.coli, and conclude that acetate has a noticeable inhibitory effect onthe maximum specific growth rate and CO₂ evolution rate constant of E.coli even at low concentrations. It is thus especially surprising todiscover that cells lacking PDH activity consume acetate, and grow well,when the preferred substrate glucose is also available; i.e., thatglucose and acetate or ethanol will serve as simultaneous substrates.

Optionally the medium additionally contains a compound from the TCAcycle such as succinate in order to fully meet the requirements of thetricarboxylic acid (TCA) cycle.

In preferred embodiment, pyruvate production in the bacterial cellsaccording to the method exceeds at least about 20 g/L, preferably about30 g/L. Preferably the yield of pyruvate from glucose (grams of pyruvateproduced per gram of glucose consumed) of at least about 0.70, morepreferably at least about 0.75. Further, the volumetric productivity ofpyruvate according to the method is at least about 1.0 g/liter-hour,more preferably at least about 1.5 g/liter-hour. Volumetric productivityis the pyruvate concentration (in g/L) divided by the fermentation timerequired to attain that concentration.

Regeneration of NAD is an important aspect in the aerobic growth of E.coli and other microorganisms. Glycolysis is possible only if NADH canbe reoxidized since NAD is a necessary participant in the oxidation ofglyceraldehyde-3-phosphate. Typically, under aerobic conditions, NADH isreoxidized by oxidative phosphorylation, a membrane-bound process whichgenerates ATP.

To our surprise, when E. coli cells lacking PDH activity were culturedaerobically, lactate was formed. The formation of lactate suggested thatthe enzyme lactate dehydrogenase (LDH), normally observed in E. colionly under anaerobic conditions, may be active in these aerobiccultures. The production of lactate in the aerobic cultures furthersuggested that the cells are not able to oxidize NADH fast enough eventhough they are grown aerobically. Because the production of pyruvateand certain derivatives of pyruvate such as diacetyl is notredox-balanced, the cells will accumulate NADH during the operation ofthe method of the invention. As a result, the pyruvate yield is expectedto improve if reoxidation of NADH is facilitated.

Overexpression of the enzyme NADH oxidase has been shown to result indiminished flux through lactate dehydrogenase (LDH) because of theremoval of this enzyme's reduced cofactor NADH. Lopez DE Felipe et al.(FEMS Microbiol. Lett. 156:15-19 (1998)) constructed an NADHoxidase-overproducing Lactococcus lactis strain by cloning theStreptococcus mutans nox-2 gene, which encodes the H₂O-forming NADHoxidase. This engineered system allowed a nisin-controlled 150 foldoverproduction of NADH oxidase resulting in decreased NADH/NAD ratiounder aerobic conditions. In the presence of flavin adenine dinucleotide(FAD), a cofactor required for NADH oxidase activity, the lactateproduction was essentially abolished. Enhancing reoxidation of NADH isthus expected to be accompanied by a reduction in the formation oflactate, which is an undesirable product.

Optionally, therefore, the bacterial cells used in the method of theinvention are further modified to increase the amount of NAD regeneratedor the rate at which NAD is regenerated. NADH can also be directlyoxidized using the nox gene which encodes NADH oxidase. E. coli cells(or other cells that lack the nox gene) that have been engineered toexpress additional NADH oxidase activity are referred to herein ashaving “added” NADH oxidase activity. It is expected that directoxidation of NADH via NADH oxidase expression will result in diminishedor possibly abolished flux toward lactate via LDH while ensuring thecells meet the demand of NAD. The presence of NADH oxidase activity isalso expected to reduce the amount of lactate formed and thereforeincrease pyruvate yield. By increasing the availability of NAD, thepresence of NADH oxidase activity is also expected to increase the rateof glucose uptake.

Therefore, in embodiments of the method used to produce products such aspyruvate or diacetyl which cause a net generation of NADH due to a redoximbalance, the bacterial cells are further optionally modified toincrease the amount of, or rate at which, NAD is regenerated from NADH.Preferably, a gene encoding NADH oxidase (nox) is introduced into thebacterial cells. Such a gene can be introduced on a plasmid or othervector, or can be chromosomally integrated into the host genome.

A process that accumulates pyruvate is also expected to accumulatebiochemicals that are a few enzymatic steps from pyruvate. For example,pyruvate is converted into alanine by a single enzyme, alaninedehydrogenase. For alanine production, therefore, the bacterial cellsused in the method of the invention preferably overexpress alaninedehydrogenase (alaD) in addition to exhibiting reduced or eliminated PDHactivity. As noted above, the bacterial cells can be further modified toreduce or eliminate the activity of pyruvate oxidase and/or PEPcarboxylase.

The production of alanine is a redox-balanced synthesis in bacterialcells, hence it is not recommended to add or increase NADH oxidaseactivity. However, alanine production can also be enhanced by reducingor eliminating the activity of lactate dehydrogenase. Thus, the methodfor producing alanine optionally utilizes bacterial cells exhibiting,compared to wild-type cells, reduced or no lactate dehydrogenaseactivity (ldh⁻).

Diacetyl, another metabolite from pyruvate, can be produced byexpressing or overexpressing acetolactate synthase in bacterial cellsexhibiting reduced or eliminated PDH activity. Conversion of pyruvate toacetolactate, which is catalyzed by acetolactate synthase, is the firststep in converting pyruvate to diacetyl. In a preferred embodiment, thebacterial cells used to produce diacetyl are E. coli cells that arepreferably further modified to exhibit added or increased acetolactatesynthase activity and/or added or increased NADH oxidase activity,and/or reduced or no activity of pyruvate oxidase and/or PEPcarboxylase, as described above.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example I Accumulation of Pyruvate in Defined Minimal Media byE. coli Mutants Lacking PDH Activity

Strains and plasmids. Strains and plasmids studied are listed inTable 1. The strains fell into two groups. Members of the first groupwere those that are blocked in their production of acetate. Thesestrains can be classified as rpoS mutants (AJW1483, CGSC5024, CGSC6159),pta mutants (CGSC5992, CGSC7237), and ack mutants (CGSC5993, CGSC7238)(CGSC: E. coli genetic stock culture. Yale University). These strainswere examined because it was hypothesized that they might accumulatepyruvate if carbon were prevented from entering the TCA cycle. Membersof the second group were those that possess mutations in genes in thePDH complex (CGSC5518—a lpd mutant, CGSC4823—an aceE mutant, andCGSC6162—an aceF mutant).

TABLE 1 Strains used Reference/ Name Genotype Source MG1655 wild-type(F⁻ λ⁻) Guyer et al., 1980 AJW1483 gal hft Δ (rpoS::Kan) Contiero etal., 2000 CGSC4823 aceE2 tyrT58(AS) trp-26 mel-1 CGSC CGSC5024 F⁺λ-rpoS390(Am) rph-1 CGSC CGSC5518 λ-lpd-1 trpA58 trpE61 CGSC CGSC5992λ-pta-39 iclR7(const) trpR80 Guest, 1979 CGSC5993 λ-gal- Guest, 19792trpA9761(Am)iclR7(const)trpR72(Am) ack-11 CGSC6159 λ-rpoS396(Am) rph-1CGSC CGSC6162 aceF10 fadR200 tyrT58(AS) adhE80 CGSC mel-1 CGSC7237 λ-Δ(his-gnd)861 hisJ701 pta-200 L_(E)Vine et al., 1980 CGSC7238 λ-Δ(his-gnd)861hisJ701ackA200 L_(E)Vine et al., 1980 CGSC: E. coli GeneticStock Center, Yale University

Media and growth conditions. An initial comparison of strains expressingendogenous PEP carboxylase was conducted using defined minimal mediamodified from (Horn et al., Appl. Microbiol. Biotechnol. 46:524-534(1996)) containing (in units of g/L): glucose, 30; KH₂PO₄, 6;(NH₄)₂HPO₄, 8; citric acid, 0.3; MgSO₄.7H₂₀, 1.5; CaCl₂.2H₂O, 0.14;Fe₂(SO₄)₃, 0.0625; H₃BO₃, 0.0038; MnCl₂.4H₂O, 0.0188; Na₂EDTA.2H₂O,0.012; CuCl₂.2H₂O, 0.0019; Na₂MoO₄.2H₂O, 0.0031; CoCl₂.6H₂O, 0.0031;Zn(CH₃COO)₂.2H₂O, 0.0099. Additionally, the media for CGSC 7237 andCGSC7238 contained 20 μg/L histidine; for CGSC4823, CGSC5518 andCGSC5993 the media contained 20 μg/L tryptophan; and for CGSC4823,CGSC5518 and CGSC6162 the media contained 1 g/L acetate. All the shakeflasks were cultured at 37° C. with 250 rpm agitation.

Analytical methods. Cell growth was monitored by measuring the opticaldensity (OD) at 600 nm (DU-650 UV-Vis spectrophotometer, BeckmanInstruments), and this value was correlated to dry cell mass. Sampleswere analyzed for glucose, pyruvate, acetate, succinate and lactatequantitatively using a previous method (M. Eiteman et al., Anal. Chim.Acia. 338: 69-75 (1997)).

Comparison of strains for growth and product formation. This exampleemployed a metabolic engineering approach for the production ofpyruvate. The strategy for generating pyruvate relied on preventing thisbiochemical intermediate from entering the TCA cycle or from beingconverted into acetate. We initially studied eleven different strains ofE. coli for acetate and pyruvate accumulation and growth rate onglucose, looking for an absence of acetate accumulation and/orrelatively high pyruvate accumulation. The initial specific growth rateswere calculated from OD measurements, and the concentrations ofby-products found after 10-20 hours of growth were used to calculateproduct yields (Table 2).

The strains having a mutation all had growth rates lower than thewild-type strain MG1655. In addition to the greatest growth rate, MG1655generated no pyruvate, and accumulated acetate to a yield of 0.11. Thethree rpoS strains each behaved differently with AJW1483 generating bothpyruvate and acetate, CGSC5024 generating neither pyruvate nor acetate,and CGSC6159 generating only acetate. Except for CGSC7237, the pta orack strains generated some acetate. CGSC7237 and CGSC7238 accumulatedsignificant pyruvate.

Of the strains with mutations in genes encoding for enzymes in thepyruvate dehydrogenase complex, the lpd strain CSGC5518 was unable togrow under the conditions studied. To our surprise, however, the aceEand aceF strains CGSC4823 and CGSC6162 accumulated the greatestconcentrations of pyruvate, resulting in pyruvate yields of 0.32 g/g and0.40 g/g, respectively.

TABLE 2 Comparison of E. coli Strains for Growth and Product Formation.Strain μ(h − 1) Y_(P/G) (g/g) Y_(A/G) (g/g) MG1655 1.57 0.00 0.11AJW1483 0.39 0.02 0.18 CGSC4823 0.91 0.32 * CGSC5024 1.17 0.00 0.00CGSC5518 0.00 — * CGSC5992 0.83 0.00 0.07 CGSC5993 0.75 0.00 0.17CGSC6159 0.72 0.00 0.16 CGSC6162 0.45 0.40 * CGSC7237 0.51 0.08 0.00CGSC7238 0.67 0.19 0.06 * media contained acetate at an initialconcentration of 1.0 g/L μ is the initial specific growth rate, Y_(P/G)is the mass pyruvate yield on glucose and Y_(A/G) is the mass acetateyield based on glucose. Results are the means of triplicate experiments.

Example II Accumulation of Pyruvate in Defined Minimal Media by E. coliMutants Lacking Both PDH and PEP Carboxylase Activity

PEP carboxylase is the enzyme that converts PEP to oxaloacetate. Thisenzyme is believed not to be necessary for growth on acetate, and wereasoned it would serve only to decrease the yield of pyruvate bysiphoning off its metabolic precursor PEP.

PEP carboxylase (ppc) mutants were generated from each of the fivestrains that accumulated pyruvate or that did not generate acetate:CGSC4823, CGSC5024, CGSC6162, CGSC7237, and CGSC7238. As noted inExample 1, CSGC4823 is an aceE mutant (genotype: aceE2 tyrT58(AS) trp-26mel-1) and CGSC6162 is an aceF mutant (aceF10 fadR200 tyrT58(AS) adhE80mel-1). As discussed in Example 1, CGSC6162 accumulates significantpyruvate even without additional genetic modification and undernonoptimized conditions.

To construct these ppc mutants, a P1 lysate from JCL 1242(λ-F-Δ(argF-lac)U169 ppc::Kan) was used to transduce each strain toKan(R) (Chao et al., Appl. Env. Microbiol. 59:4261-4265 (1993)). Becausethese ppc strains lacked the anaplerotic enzyme PEP carboxylase, themedia as described in Example I was supplemented additionally with bothacetate and succinate. The acetate and succinate were supplied atinitial concentrations of either 0.62 g/L or 4.0 g/L each. The purposeof this study was to determine the effect of ppc deletion on growth rateand pyruvate formation, and the results are shown in Table 3.

TABLE 3 Comparison of E. coli ppc strains for growth rate. InitialSuccinate and Acetate μ Y_(P/G) Q_(P) Strain (g/L) (h − 1) (g/g) (g/Lh)CGSC4823 Δppc 0.62 0.03 0.14 0.03 4.0 0.04 0.08 0.03 CGSC5024 Δppc 0.620.03 0.07 0.02 4.0 0.04 0.00 0.00 CGSC6162 Δppc 0.62 0.04 0.76 0.28 4.00.13 0.72 0.15 CGSC7237 Δppc 0.62 0.12 0.06 0.01 4.0 0.05 0.00 0.00CGSC7238 Δppc 0.62 0.11 0.11 0.03 4.0 0.05 0.00 0.00 μ is the initialspecific growth rate, Y_(P/G) is the mass pyruvate yield based onglucose and Q_(P) is the volumetric productivity of pyruvate.

Deletion of the ppc gene resulted in slower growth rates and generallymore pyruvate accumulation than the strains with ppc gene. Except forCGSC6162 Δppc, the maximum cell-mass produced with 4.0 g/L initialconcentrations was considerably higher (˜2 times) than with 0.62 g/L.

Strains CGSC5024 Δppc, CGSC7237 Δppc, and CGSC7238 Δppc did not consumeacetate when grown on 0.62 g/L initial concentrations and in factgenerated additional acetate once succinate was consumed. These threestrains also did not accumulate pyruvate with 4.0 g/L initialconcentrations and had the three lowest pyruvate yields with 0.62 g/Linitial concentrations.

CGSC4823 Δppc and CGSC6162 Δppc accumulated significant pyruvate underboth initial conditions. CGSC4823 Δppc produced less pyruvate thanCGSC6162 Δppc and also accumulated acetate. CGSC6162 Δppc did notaccumulate acetate. A small amount of lactate generation was alsoobserved with strains CGSC4823 Δppc and CGSC6162 Δppc. From this studyCGSC4823 Δppc and CGSC6162 Δppc were selected for the fermenter studies.

Example III Accumulation of Pyruvate in Rich Medium by E. coli MutantsEither Lacking PDH Activity or Lacking Both PDH and PEP CarboxylaseActivity

Strains and plasmids. The strains studied were among those listed inTable 1 in Example I. The strains included an rpoS mutant (CGSC5024),pta mutant (CGSC7237), ack mutant (CGSC7238) and a representative mutantfor each of the three genes of the PDH complex, aceE, aceF and lpd(CGSC4823, CGSC6162, CGSC5518, respectively). A Δppc mutation wasintroduced into each of these strains as described in Example II.

Media and growth conditions. An initial comparison of strains wasconducted using 100 mL of media containing (g/L): glucose, 10.0; aceticacid, 3.0; succinic acid, 6.0; yeast extract, 2.5; tryptone, 5.0;KH₂PO₄, 6.0; (NH₄)₂HPO₄, 8.0; citric acid, 0.3; MgSO₄.7H₂O, 1.5;CaCl₂.2H₂O, 0.14; Fe₂(SO₄)₃, 0.0625; H₃BO₃, 0.0038; MnCl₂.4H₂O, 0.0188;Na₂EDTA.2H₂O, 0.012; CuCl₂.2H₂O, 0.0019; Na₂MoO₄.2H₂O, 0.0031;CoCl₂.6H₂O, 0.0031; Zn(CH₃COO)₂.2H₂O, 0.0099. All the 500 mL shakeflasks were cultured in duplicate at 37° C. with 250 rpm agitation andinitial pH of 7.0.

Subsequent studies of selected strains were conducted incomputer-controlled fermentations of 1.5 L volume carried out in 2.5 Lfermenters (Bioflow III, New Brunswick Scientific Co., Edison, N.J.).Unless otherwise stated, the temperature was maintained at 37° C.,agitation at 750 rpm, sterile filtered air was sparged at a rate of 1.5L/min, and 20% NaOH and 20% HCl were used to control pH. The level ofaeration ensured that the dissolved oxygen never fell below 30% ofsaturation for any of the fermentations. Samples were taken periodicallyand stored at −20° C. for subsequent analysis. The medium was identicalto that described above except for initial concentration of 40 g/Lglucose. For fed batch fermentations, glucose concentration wasmaintained at 3 g/L by automatic feeding of a 600 g/L glucose solution(YSI, Yellow Springs, Ohio).

Comparison of strains for growth and product formation. This exampleemployed a metabolic engineering approach for the production ofpyruvate. The strategy for generating pyruvate relied on preventing thisbiochemical intermediate from entering the TCA cycle or from beingconverted into acetate. Therefore, we initially studied six strains ofE. coli and their corresponding ppc mutants for growth and acetate andpyruvate accumulation using a medium containing 10 g/L glucose. For allthese strains, the glucose was exhausted in 7-10 hours. Table 4summarizes the results of these duplicate shake flask fermentations,with pyruvate and acetate yields calculated at the time that glucose wasexhausted.

TABLE 4 Pyruvate and acetate mass yields in strains of Escherichia coli.Strain Max. OD Y_(P/G) Y_(A/G) CGSC4823 8.1 0.54 −0.01* CGSC4823 Δppc5.1 0.097 0.19 CGSC5024 8.8 0.085 0.34 CGSC5024 Δppc 4.7 0.12 0.28CGSC5518 6.1 0.31 0.11 CGSC5518 Δppc 8.0 0.083 0.30 CGSC6162 6.8 0.470.030 CGSC6162 Δppc 6.9 0.41 0.029 CGSC7237 6.7 0.34 0.024 CGSC7237 Δppc5.5 0.11 0.30 CGSC7238 7.0 0.25 0.17 CGSC7238 Δppc 5.3 0.10 0.23Y_(P/G): pyruvate generated/glucose consumed (g/g) Y_(A/G): acetategenerated/glucose consumed (g/g) *For this growth condition and timeinterval, the organism consumed acetate

Strains with alterations in acetate synthesis included CGSC5024 (rpoS),CGSC7237 (pta) and CGSC7238 (ack). Of these CGSC7237 and CGSC7238accumulated greater than 25% (mass yield) pyruvate, but only CGSC7237generated less than 15% acetate. Introduction of the ppc mutationincreased pyruvate accumulation only with CGSC5024, the lowest pyruvateproducer. With CGSC7237 a ppc mutation increased acetate yieldtwelve-fold and decreased pyruvate yield three-fold. For all threestrains a deletion in ppc substantially reduced the maximum cellconcentration and hence cell yield. Strains with mutations in the PDHcomplex included CGSC4823 (aceE), CGSC6162 (aceF) and CGSC5518 (lpd).Each of these strains initially consumed acetate, accumulatedsignificant pyruvate and began accumulating acetate after 2-4 hours. Bythe time glucose was exhausted, only CGSC4823 had a small netconsumption of acetate. Introduction of the ppc mutation decreasedpyruvate yield for all three strains, and increased acetate yield in theCGSC4823 and CGSC5518. A deletion in ppc increased cell yield forCGSC5518, decreased the cell yield for CGSC4823, and had no effect oncell yield in CGSC6162.

Example IV Batch Fermentation Studies on CGSC4823, CGSC4823 Δppc,CGSC6162 and CGSC6162 Δppc

CGSC4823 Δppc and CGSC6162 Δppc, along with their parent strain(CGSC4823 and CGSC6162) were grown at 1.5 L (initial volume) in a 2.5 Lfermenter (New Brunswick Scientific Instruments, NJ) at 37° C. and 750rpm with 1.5 L/min constant air flowrate using three different media(Media A, Media B and Media C). The modified Horn medium (Example I) wasagain used but with different initial concentrations of various carbonsources. Medium A contained 20 g/L glucose, 1 g/L acetate and 2 g/Lsuccinate. Medium B contained 40 g/L glucose, 3 g/L acetate and 6 g/Lsuccinate. Medium C contained 40 g/L glucose, 3 g/L acetate, 6 g/Lsuccinate, 2.5 g/L yeast extract and 5 g/L tryptone. When the glucoseconcentration decreased to 3.0 g/L, it was controlled at thisconcentration by the automatic feeding of a 600 g/L glucose solutionusing an on-line glucose analyzer (YSI Instruments, OH). Samples weretaken periodically during growth and stored at −20° C. for subsequentanalysis. Analytical methods were as in Example I.

The results are shown in Table 5. Cells generally grew to their lowestcell mass in media A because of the lower concentrations of acetate andsuccinate in the media. Supplementing the media with yeast extract andtryptone (Media C) reduced the lag phases and generally increased thegrowth rates. FIG. 2 shows the products of fermentations using Media Cand the strains (a) CGSC4823, (b) CGSC4823 Δppc, (c) CGSC6162 and (d)CGSC6162 Δppc, respectively. In these fermentations the pyruvate yieldwas greater than 0.50 except for CGSC4823 Δppc. Very small amounts oflactate were observed in CGSC4823 Δppc and CGSC6162 Δppc. From thisstudy, CGSC6162 and CGSC6162 Δppc were selected for further fed-batchfermentation studies using Media C.

TABLE 5 Comparison of E. coli strains for growth rate and productformation when grown on different media. Media μ Max. Pyr. Y_(P/G)Strain (g/Lh) (h⁻¹) Max. OD (g/L) (g/g) Q_(P) CGSC4823 A 0.18 4.1 9.80.58 0.28 B 0.10 5.0 17.0 0.46 0.29 C 0.20 11.0 20.0 0.52 1.10 CGSC4823Δppc A 0.08 2.2 10.0 0.63 0.10 B 0.04 7.5 16.0 0.41 0.29 C 0.07 22.3 1.10.03 0.06 CGSC6162 A 0.17 7.2 11.0 0.56 0.69 B 0.11 13.0 18.0 0.53 0.56C 0.11 10.0 23.0 0.63 1.50 CGSC6162 Δppc A 0.03 0.7 3.7 0.91 0.09 B 0.020.4 0.4 — — C 0.19 11.0 24.0 0.64 1.10 Media A: 20 g/L glucose, 1 g/Lacetate, 2 g/L succinate Media B: 40 g/L glucose, 3 g/L acetate, 6 g/Lsuccinate Media C: 40 g/L glucose, 3 g/L acetate, 6 g/L succinate, 2.5g/L yeast extract, 5 g/L tryptone μ is the initial specific growth rate,Y_(P/G) is the mass pyruvate yield based on glucose and Q_(P) is thevolumetric productivity of pyruvate.

Example V Fed-Batch Fermentation Studies on CGSC6162 and CGSC6162Δppc toStudy the Effect of pH

We next conducted fed-batch fermentations with CGSC6162 and CGSC6162Δppc to study the affect of pH on pyruvate accumulation. Thefermentations again commenced at a pH of 7.0 with a glucoseconcentration of 40 g/L. After 12 hours of growth, the pH was shifted(over the course of about 30 minutes) to the desired constant pH. Whenthe glucose concentration reached 3.0 g/L (at 16-18 hours), glucose wasmaintained at that concentration until the fermentations terminated at36 hours. Table 6 shows the mean specific rates of glucose consumptionand formation for the three products at the pH levels studied over thetime interval of 12 hours to 36 hours. The results include the specificactivities of LDH and POX at 20 hours.

The pH had a significant effect on the CGSC6162 fermentations. At thelowest pH (6.0), glucose consumption was low, and acetate was theexclusive product (with a mass yield of 67%, essentially the theoreticalmaximum). Also, the activity of POX was relatively high. At the otherthree levels of pH (6.5, 7.0, 7.5), additional acetate did not form,pyruvate was the primary product, but lactate formation was alsosignificant. The activity of POX was 3-4 times lower than observed at apH of 6.0. For all CGSC6162 fermentations during the initial phase (pH7.0 until 12 hours), pyruvate mass yield was approximately 70%.Thereafter the pyruvate yield decreased with time. For example, duringthe interval 12-20 hours, the yields were 59% (pH of 6.5), 72% (7.0),52% (7.5), while during the interval 20-28 hours the yields were 25%(6.5), 32% (7.0), 38% (7.5). In the pH range of 6.5-7.5, lactateaccumulated only after the acetate was exhausted. The maximum pyruvateconcentration achieved was about 35 g/L for the fermentationscontinuously at a pH of 7.0.

For the CGSC6162 Δppc fermentations at the lowest pH of 6.0, thepyruvate that had been formed during the first 12 hours at a pH of 7.0was partly consumed. Acetate was the exclusive product, and the POXactivity was high. At pH values of 6.5 and 7.0, acetate was stillformed, but pyruvate was the principal product and POX activity wasabout half that observed at a pH of 6.0. At a pH of 6.5 and 7.0, the POXactivity was about two times greater in CGSC6162 Δppc than in CGSC6162.For the CGSC6162 Δppc fermentations during the initial phase (pH 7.0until 12 hours), pyruvate mass yield was about 75% and again, thepyruvate yield decreased thereafter during the course of thefermentations. The LDH activity was low in all cases, did not correlatewith lactate formation, and did not appear to follow any trend with pH.

TABLE 6 Specific rates of glucose consumption and pyruvate, acetate andlactate generation during fed-batch fermentations of CGSC6162 andCGSC6162 Δppc at different levels of pH. First 12.0 hours of growthoccurred at a pH of 7.0, after which time the pH was gradually changedto indicated pH for an additional 24 hours and the rates recorded.Enzyme activities at 20 hours are in U/mg protein. Strain pH q_(G) q_(P)q_(A) q_(L) LDH POX CGSC6162 6.0 0.34 0 0.23 0 0.08 2.25 6.5 0.94 0.27 00.26 0.08 0.51 7.0 0.89 0.34 0 0.18 0.06 0.51 7.5 0.80 0.31 0 0.21 0.090.77 CGSC6162 Δppc 6.0 0.32 −0.15* 0.30 0 0.08 1.92 6.5 0.57 0.21 0.13 00.01 1.04 7.0 0.60 0.32 0.06 0.05 0.00 1.07 q: specific rate offormation/consumption during the time interval of 12 hours to 36 hours(g compound/g cells hour) *For this growth condition and time interval,the organism consumed this compound

Several remarkable results are shown in FIG. 3. E. coli CGSC6162 didindeed simultaneously consume acetate and glucose. Interestingly, duringthe initial portion of these fermentations, these strains consumed allthe acetate supplied, and the maximum concentrations of pyruvateoccurred when the acetate concentration reached zero. Cell growth ceasedwhen the acetate became depleted 8 hours after inoculation,demonstrating that this substrate was necessary for cell growth. Eventhough acetate was depleted in less than 10 hours, the cells generatedwell over 30 g/L pyruvate, and the mass yield of pyruvate from glucosewas about 0.70 during the first 20 hours. The volumetric productivityduring this time interval of low cell density was over 1.5 g/L hour.

After 20 hours, lactate surprisingly appeared as a co-product withacetate, with the concentration of pyruvate diminishing. Lactate (andNAD) generation from pyruvate (and NADH) by lactate dehydrogenase isknown to be used by E. coli as a means to balance the cofactors NADH andNAD (Gokarn et al., Appl. Env. Microbiol. 66:1844-1850 (2000)). Ittherefore is particularly interesting that lactate was synthesizedduring this aerobic fermentation in which NADH could generate energy forthe cell, as it suggests that NAD could not be regenerated from NADHquickly enough via oxidative phosphorylation to meet the demand ofglucose uptake through the EMP pathway.

An interesting observation is that the generation of carbon dioxide wasmuch lower than commonly observed in aerobic fermentations, which can beexplained as follows. During the production of pyruvate which appears tohave come from 70% of the glucose in our study, no carbon dioxide isgenerated from the conversion of glucose to pyruvate. One mole of carbondioxide is generated from each mole of carbon entering the pentosephosphate pathway (likely less than 15% of the total glucose), and onlya small quantity of carbon dioxide will be generated from theconsumption of acetate through the TCA cycle, primarily for toward thesynthesis of biomass.

The results demonstrate that a pyruvate yield exceeding 0.75 wasroutinely obtained during the first 12 hours of these fermentations.Moreover, the results demonstrate that changing the pH from 7.0 after 12hours did not improve the pyruvate yield. Switching to a pH of 6.0 forboth strains resulted in the consumption of pyruvate and generation ofacetate. This utilization of pyruvate did not result in an increase inthe cell mass concentration. FIG. 3( a) shows the fermentation usingCGSC6162 at a pH of 6.0. FIG. 3( b) shows the fermentation usingCGSC6162 at a pH of 7.0. In cases in which the pH remained at 7.0, thevolumetric productivity of pyruvate at the time acetate was depleted wasover 1.5 g/Lh. This is the first report of a viable industrial processfor the production of pyruvate in E. coli, something that was heretoforethought impossible because of the complexities and interdependencies ofthe interrelated metabolic pathways that stem from the pyruvate node.

Example VI Fed-Batch Fermentation Studies on CGSC6162 and CGSC6162Δppcto Study the Effect of Temperature

Fed-batch fermentations of CGSC6162 and CGSC6162 Δppc were also studiedat three different temperatures (32° C., 37° C. and 42° C.) at pH 7.0for 36 hours. Pyruvate and lactate yields were calculated for two timeintervals (0-20 hours; and 20-36 hours), and the results are shown inTable 7.

CGSC6162 did not accumulate acetate at any temperature (FIG. 4 shows a42° C. fermentation). Lactate and pyruvate production were stronglyinfluenced by temperature. The initial rate of pyruvate formation wasgreatest at 42° C., with 21-25 g/L accumulating in 12 hours. However,after about 12 hours at this temperature, the cell density decreased,and CGSC6162 accumulated lactate instead of pyruvate. Thus, the greatestpyruvate concentrations and lowest lactate concentrations over thecourse of 36 hours were achieved when the fermentation temperature wasmaintained at 32° C.

Although initially CGSC6162 Δppc consumed acetate, this straineventually accumulated acetate at all temperatures (FIG. 5 shows anexample CGSC6162 Δppc fermentation at 32° C.). However, the accumulationof acetate was greater and commenced sooner at higher temperature.Specifically, at 42° C. acetate began accumulating at about 8 hours, at37° C. acetate accumulation began at 12-16 hours, and at 32° C. acetatebegan accumulating after 20 hours. Similar to CGSC6162, with CGSC6162Δppc lactate accumulation began after 12 hours for all temperatures. Therate of lactate production was again strongly temperature dependent,with higher temperature favoring lactate. This strain also achieved itsmaximum pyruvate concentration at 32° C.

Table 7 shows the specific activities of LDH and POX at 20 hours for thetwo strains at the three different temperatures. In all cases the LDHactivity was low and did not follow a trend. POX activity tended toincrease with increasing temperature in both CGSC6162 and CGSC6162 Δppc.Furthermore, POX activity was about twice as great in CGSC6162 Δppc thanin CGSC6162 at 37° C. and 42° C. As the data indicates, the productionof pyruvate may further be increased by deleting the pox gene.

TABLE 7 Yields of pyruvate and lactate during fed-batch fermentations ofCGSC6162 and CGSC6162 Δppc at different controlled temperatures. Yieldsare calculated during two time intervals (0-20 hours and 20-36 hours).Enzyme activities at 20 hours are in U/mg protein. Temp Maximum PyruvateY_(P/G) Y_(L/G) Strain (° C.) Concentration (g/L) 0-20 h 20-36 h 0-20 h20-36 h LDH POX CGSC6162 32 37 0.73 0.47 0.03 0.07 0.04 0.57 37 36 0.670.25 0.08 0.20 0.06 0.51 42 32 0.67 0.16 0.01 0.58 0.07 0.80 CGSC6162Δppc 32 35 0.70 0.60 0.01 0.07 0.01 0.45 37 35 0.74 0.41 0.02 0.11 0.001.07 42 29 0.57 0.25 0.06 0.60 0.01 1.58 Y_(P/G): pyruvategenerated/glucose consumed (g/g) Y_(L/G): lactate generate/glucoseconsumed (g/g)

Example VII Overexpression of NADH Oxidase to Produce Pyruvate

The accumulation of small amounts of lactate during highly aerobicconditions (see Example V and VI) suggests that NADH is not beingconverted into NAD to keep pace with the demand of glycolysis. It wouldbe of little benefit to delete lactate dehydrogenase activity as a meansof producing pyruvate and avoiding lactate generation, as this does notaddress what appears to be the underlying cause, namely, the conversionof NADH to NAD. Furthermore, cells do not appear to be limited in ATPgeneration, since the NADH is being consumed toward lactate formationrather than via oxidative phosphorylation.

One way to enhance the regeneration of NAD is by introducing additionalNADH oxidase activity into the strains. The nox gene encodes NADHoxidase which converts NADH and oxygen directly into NAD and water(without the generation of ATP). Known nox genes encoding for NADHoxidase include those from Streptococcus pneumoniae, S. faecalis andSaccharomyces cerevisiae. Lopez de Felipe et al. (FEMS Microbiol. Lett.156:15-19 (1998)) have previously used NADH oxidase overexpression inLactococcus lactis to significantly decrease the NADH/NAD ratio andreduce lactate synthesis. We have constructed a pTrc99A-nox plasmidwhich overproduces NADH oxidase from S. pneumoniae. This plasmid, whichis inducible by the addition of IPTG, can be used to transform PDH andPDH+PEP carboxylase mutant strains to enhance the production ofpyruvate.

Example VIII Enhanced Production of Diacetyl

If pyruvate can accumulate significantly in cells, then biochemicalderivatives of pyruvate might also accumulate in these cells.Diacetyl(2,3-butanedione), with a vapor pressure similar to ethanol, isa constituent of food and fruit aromas and is the main constituent of“butter aroma.” As shown in FIG. 1, the synthesis of diacetyl firstinvolves the conversion of pyruvate to acetolactate by the enzymeacetolactate synthase. Diacetyl can be synthesized from pyruvate by atwo step process involving 1) the conversion of pyruvate to acetolactateby the enzyme acetolactate synthase and 2) the chemicaloxidation/decomposition of acetolactate to diacetyl. These twoadditional pathways are shown in FIG. 1.

Plasmid pAAA215 overproduces acetolactate synthase from Bacillussubtilis (Aristidou et al., Biotechnol. Bioeng. 44:944-951 (1994)). Thesynthesis of this enzyme appears to be induced by cell growth and itsactivity is stimulated by the presence of acetate (Holtzclaw et al., J.Bacteriol. 121:917-922 (1975)). The product acetolactate itself ischemically unstable, being oxidized (by oxygen) to diacetyl in thepresence of metal ions such as Fe³⁺. Two competing biochemical reactionscan also occur. In some organisms, acetolactate decarboxylase catalyzesthe decarboxylation of acetolactate under oxygen limited conditions toacetoin. The chemical oxidation of acetolactate appears to be favored ata pH of about 5, while the enzymatic decarboxylation of acetolactateappears to be favored at a pH of about 6.5. The presence of acetolactatedecarboxylase activity in E. coli has not been established. A secondcompeting reaction involves the enzyme diacetyl reductase, whoseactivity has been observed in E. coli, which directly converts diacetylto acetoin. This reaction has a pH optimum of about 7.0, and requiresNADH or NADPH, the former being more active with the latter.Interestingly, acetate has been shown to inhibit the activity of thisenzyme, 20 mM decreasing the activity by 35% (Ui, Agr. Biolog. Chem.51:1447-1448 (1987)). Moreover, under highly oxygenated conditions, NADHand NADPH will normally be less prevalent than their oxidized analogues.The pH optima, the effects of acetate and oxygen, and the prospects foradding other chemical catalysts or inhibitors would suggest that afermentation process to accumulate diacetyl is feasible under thegeneral conditions we have previously observed for pyruvate generation.

To accomplish, this, the “best” strains as identified in Example I aretransformed with the pAAA215 plasmid that overproduces acetolactatesynthase from B. subtilis (Aristidou et al., Biolechnol. Bioeng.44:944-951 (1994)). The plasmid pTrc99A-nox, which overproduces NADHoxidase, can also be transformed into these strains. Because theycontain compatible replicons, both the pTrc99A-nox plasmid whichoverproduces NADH oxidase and the pAAA215 plasmid which overproducesacetolactate synthase can be introduced into the same cell. ThepTrc99A-nox plasmid uses the colE1 replicon and is selected for usingampicillin while the pAAA215 plasmid uses the P15A replicon and isselected for using tetracycline. Numerous researchers have constructeddual plasmid strains like this where the first plasmid contained thecolE1 replicon and the second plasmid contained the P15A replicon. Theresult is the construction of at least one strain with a PDH mutationand enhanced acetolactate synthase activity, and also a strain withadditional increased NADH oxide activity.

The chemical production of diacetyl is promoted by oxidation. Theconversion of diacetyl to acetoin by the undesirable enzymatic reaction(diacetyl reductase) uses NADPH or NADH as a cofactor with the formerbeing preferred. We would expect a highly oxygenated environment toprevent this undesirable reaction. It does not appear feasible toperform a gene “knock out” of diacetyl reductase as evidence suggeststhat this reaction is carried out inadvertently (i.e., nonspecifically)by one or more general reductase enzymes. The presence of NADH oxidaseshould facilitate diacetyl production because, like pyruvate generationitself, a greater rate of NADH oxidation would tend to increase the rateof glucose uptake and reduce the availability of NADH/NADPH for sidereactions.

Example IX Enhanced Production of Alanine

Alanine can be synthesized from pyruvate in a single reaction step bythe enzyme alanine dehydrogenase. L-alanine is generally produced by anenzymatic process in which L-aspartic acid is enzymaticallydecarboxylated (Ichiro et al., U.S. Pat. No. 3,458,400 (1969)). E. colihas activity in a racemase, which would convert the L-alanine producedin any process to D-alanine, resulting in a DL-alanine product. Althoughultimately the activity of alanine racemase can be abolished to yieldexclusively the L-alanine product, this example focuses on theproduction of the racemic mixture of alanine.

In order to test whether the same approach could be used to generatederivatives of pyruvate, such as alanine, we transformed CGSC6162 withthe pTrc99A-alaD plasmid that we constructed which overproduces alaninedehydrogenase from Bacillus sphaericus (Ohashima et al., Eur. J.Biochem. 100:29-39 (1979)). Alanine dehydrogenase is an enzyme thatconverts pyruvate to alanine. This strain was grown on media containing25 g/L glucose, 3.0 g/L yeast extract, 6.0 g/L tryptone, 2.7 g/Lsuccinate, 1 g/L acetate, 1 mg/L biotin, 1 mg/L thiamine HCl, 15 mg/LCaCl₂2H₂O, 5.875 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 6 g/L NH₄Cl and 0.25 g/LMgSO₄7H₂O. Continuous constant agitation (1000 rpm) and air flowrate(1.0 L/min) were used.

FIG. 6 shows the results, with alanine accumulating to 3.3 g/L. Alanineaccumulated to this level even though the nitrogen concentration (as N)was less than 2 g/L in the initial media. This nitrogen would berequired both for cell growth (about 12% N) and alanine synthesis (about15% N). The activity of alanine dehydrogenase was 0.04 U/mg at 8 hoursand 0.06 U/mg at 14 hours.

The formation of alanine consumes NADH in the final step via alaninedehydrogenase. Considering that the cell would otherwise generate ATPfrom the oxidative phosphorylation of NADH, and full aeration was usedin our study with dissolved oxygen concentration always above 75%saturation, this level of accumulation is remarkable.

Interestingly, significant pyruvate still accumulated (10 g/L). Thissuggests that alanine production was limited due to a non-optimal amountof alanine dehydrogenase. The activity of alanine dehydrogenase in oursystem can be increased by recloning the B. sphaericus alaD gene into apTrc99A derivative that we have constructed which expresses genes at alevel 3-10 times higher than is obtainable in the original pTrc99Avector.

The biochemistry of alanine production has a crucial difference from thebiochemistry of pyruvate production. The production of pyruvate fromglucose generates 2 moles of NADH per mole of glucose, and should beconducted with high oxygenation so the NADH produced can be converted toATP by oxidative phosphorylation. Moreover, oxygen limitation in thiscase could lead to activation of pyruvate formate lyase and subsequentreduction of pyruvate through fermentative regeneration of NAD. Incontrast, the overall production of alanine from glucose does notgenerate NADH due to the final step from pyruvate to alanine. Sinceoxygenation affects the NADH/NAD balance, the availability of oxygenshould be have a significant impact on alanine production. We expectthat reduction of oxygen availability should improve alanine productionuntil a level is reached where the fermentative enzymes such as lactatedehydrogenase and pyruvate formate lyase are induced. The oxidation“state” of the system can be most readily monitored by dissolved oxygenconcentration or by the culture's redox potential.

Note that because alanine dehydrogenase consumes NADH, NADH productionis balanced during the conversion of glucose to alanine. It wouldtherefore be unnecessary, and indeed undesirable, to overexpress NADHoxidase to enhance the production of the pyruvate derivative alanine.

Example X Enhanced Production of Alanine in a Lactate DehydrogenaseMutant

Lactate dehydrogenase converts pyruvate to lactate and thus competeswith alanine dehydrogenase, which converts pyruvate to alanine. Thelactate dehydrogenase and alanine dehydrogenase enzymes both usepyruvate and NADH as substrates; therefore, if native lactatedehydrogenase is present during a fermentation in which alanine is thedesired product, the lactate dehydrogenase could undesirably competewith alanine dehydrogenase.

In order to prevent lactate dehydrogenase from possibly competing withalanine dehydrogenase in the generation of the pyruvate derivativealanine, we constructed an ldhA deletion mutant of CGSC6162. In E. coli,ldhA is the gene that encodes lactate dehydrogenase. We also furtherimproved the alanine production process regarding oxygenation bycurtailing agitation during the course of the fermentation as suggestedin Example IX. The ldhA::Kan deletion mutant from the E. coli strainNZN111 (Bunch et al., Microbiology, 143:187-195 (1997)) was introducedinto CGSC6162 by P1 phage transduction. We transformed this CGSC6162ldhA deletion mutant with the pTrc99A-alaD plasmid that we constructedwhich overproduces alanine dehydrogenase from Bacillus sphaericus.

CGSC6162 ldhA::Kan pTrc99A-alaD cells were grown in a BioFlow 2000fermenter (New Brunswick Scientific Co., New Brunswick, M.J.), with 1.5L of media containing 40.0 g/L glucose, 6.0 g/L succinic acid, 3.0 g/Lacetic acid, 10 g/L tryptone, 2.5 g/L yeast extract, 3.0 g/L KH₂PO₄, 6.0g/L NaH₂PO₄, 6.0 NH₄Cl, 0.14 g/L CaCl₂.2H₂O and 0.25 g/L MgSO₄.7H₂O, and100 mg/L ampicillin. The fermenter was operated at 37° C., a pH of 7.0,with 1000 rpm agitation and 1.0 L/min air flow. After 4.0 hours ofgrowth in the fermenter, IPTG was added to a final concentration of 1.0mM. At 11.0 hours of growth in the fermenter, the agitation was reducedto 250 rpm. At 15.0 hours of growth in the fermenter, an additional 110mL volume of solution was added to the fermenter containing 30 g glucoseand 7.5 g NH₄Cl.

FIG. 7 shows the results, with alanine accumulating to 12 g/L. Theseresults show that the deletion of ldhA in CGSC6162 does notdeleteriously impact cell growth. Furthermore, these results demonstratethat additional improvement in the production of pyruvate derivativessuch as alanine can be attained both by genetic means (ldhA mutation)and by process modifications (optimal oxygenation).

Increased Yield with Increased Ammonium

By altering growth conditions and supplying additional ammonium 10chloride, the yield of alanine increased to 32 g/liter. See M. Lee etal., Appl. Microbiol. Biotechnology 65: 56-60 (2004).

Specifically, cells of CGSC6162 ldhA pTrc99A-alaD were first grown at 20mL volume in an agitated screw top test tube with media composed of (perliter) 15.0 g glucose, 3.0 g acetic acid, 6.0 g succinic acid, 2.5 gtryptone, 2.5 g NaCl, and 1.25 g yeast extract. After 3 hours of growth10 mL was used to inoculate 100 mL of media in a 250 mL baffled shakeflask composed of (per liter) 15.0 g glucose, 3.0 g acetic acid, 6.0 gsuccinic acid, 10.0 g tryptone, 2.5 g yeast extract, 3.0 g KH₂PO4, 6.0 gNa₂HPO4, 6.0 g NH₄Cl, 0.14 g CaCl₂.H₂O, and 0.25 g MgSO₄.7H₂O. Cellswere grown at 250 rpm (19 mm radius of orbit) for 6 hours and then usedto inoculate a fermenter of the same composition as the shake flaskexcept 40 g/liter glucose. Fermentations of 1.5 liter initial volumewere conducted using a BioFlow 2000 (New Brunswick Scientific Company,New Brunswick, N.J.). Air was supplied continuously at 1.0 liter/minute.After 3-4 hours of growth in the fermenter, 1.0 mMisopropyl-β-D-thiogalactopyranoside (IPTG) was added for gene induction.During the first 11 hours of fermentation, the agitation was 1000 rpm, arate which insured that the dissolved oxygen remained above 20% ofsaturation. At 11 hours the alanine production phase was initiated byreducing the agitation rate to a lower constant value as described inthe text. Oxygen mass transfer coefficients (k_(L)a) for eachexperimental agitation rate were determined in a separate experimentusing the static sparging method (W. S. Wise, J. Gen. Microbiol. 5:167-177 (1951)) with identical media and fermenter system. At 15 hours,additional glucose and NH₄Cl was added as described in the text toreplenish these components that had been consumed for the generation ofcell mass and alanine. All media contained 100 mg/liter ampicillin andwere carried out at 37° C. and a pH of 7.0 controlled throughout thefermentations.

In studies on the effect of k_(L)a on alanine accumulation, a consistentresult was that alanine generation occurred at the highest rateimmediately following the addition of glucose and NH₄Cl at 15 hours. Inorder to determine whether NH₄Cl was limiting the conversion of pyruvateto alanine via alanine dehydrogenase, we determined the ammonium ionconcentration at the end of these fermentations, and found the ammoniumconcentration to be a minimum of 20 mmol/liter. We then repeated thosefermentations with the lowest value of k_(L)a of 7 hour⁻¹. In this case,however, we provided three times the NH₄Cl (22.5 g) with the 30 gglucose at 15 hours and then both materials again at 23 hours. Theresulting rate of alanine production was consistently above 2.0g/liter-hour between 15 hours and 27 hours (FIG. 8), significantlygreater than previously when less NH₄Cl was added. In duplicateexperiments, the alanine concentration reached 32 g/liter in 27 hour,but the alanine concentration did not increase further regardless ofwhether additional glucose and NH₄Cl was added. The overall alanineyield on glucose averaged 0.63 g/g, and the alanine yield on glucoseafter 15 hours averaged 0.81 g/g.

Example XI Reduction of Pyruvate Oxidase Activity

An enzyme that can assimilate pyruvate is pyruvate oxidase. We observedsignificant pyruvate oxidase activity (over 1.00 IU/mg protein) afteracetate was depleted in all the fed-batch fermentations operated atvarious levels of pH (Example V). These results suggest that reducing oreliminating pyruvate oxidase activity would prevent a portion of thepyruvate generated from being lost.

One approach is to knock out the poxB gene in E. coli expressingpyruvate oxidase. These strains are expected to grow and accumulatepyruvate at higher levels under the previously tested conditions.

The complete disclosures of all patents, patent applications includingprovisional patent applications, and publications, and electronicallyavailable material (e.g., GenBank and Protein Data Bank amino acid andnucleotide sequence submissions) cited herein are incorporated byreference. The foregoing detailed description and examples have beenprovided for clarity of understanding only. No unnecessary limitationsare to be understood therefrom. The invention is not limited to theexact details shown and described; many variations will be apparent toone skilled in the art and are intended to be included within theinvention defined by the claims.

1. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing a bacterial cell exhibiting reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell; and culturing the bacterial cell in the presence of glucose and acetate under conditions and for a time effective to accumulate the metabolite to a concentration of at least about 3.3 g/L.
 2. The method of claim 1 wherein the metabolite comprises pyruvate or diacetyl.
 3. The method of claim 1 wherein the bacterial cell further exhibits added or increased NADH oxidase activity compared to a wild-type bacterial cell.
 4. The method of claim 1 wherein the metabolite comprises alanine.
 5. The method of claim 1 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 6. The method of claim 5 wherein the additional carbon source comprises succinate.
 7. The method of claim 1 wherein the metabolite comprises pyruvate.
 8. The method of claim 7 wherein pyruvate is produced in an amount of at least about 30 g/L.
 9. The method of claim 7 wherein the pyruvate yield is at least about 0.70.
 10. The method of claim 1 wherein the PDH activity in the bacterial cell is undetectable.
 11. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing an E. coli cell exhibiting reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type E. coli cell; and culturing the E. coli cell in the presence of glucose and acetate to yield the metabolite.
 12. The method of claim 11 wherein the metabolite comprises pyruvate or diacetyl.
 13. The method of claim 11 wherein the E. coli cell further exhibits added NADH oxidase activity.
 14. The method of claim 11 wherein the metabolite comprises alanine.
 15. The method of claim 11 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the E. coli cell.
 16. The method of claim 15 wherein the additional carbon source comprises succinate.
 17. The method of claim 11 wherein the metabolite comprises pyruvate.
 18. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing a bacterial cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes compared to a wild-type bacterial cell, and (b) reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a wild-type bacterial cell; and culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.
 19. The method of claim 18 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 20. The method of claim 19 wherein the additional carbon source comprises succinate.
 21. The method of claim 19 wherein the metabolite comprises pyruvate.
 22. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing a bacterial cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes compared to a wild-type bacterial cell, and (b) added or increased NADH oxidase activity; and culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.
 23. The method of claim 22 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 24. The method of claim 23 wherein the additional carbon source comprises succinate.
 25. The method of claim 23 wherein the metabolite comprises pyruvate.
 26. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing a bacterial cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase(PDH) complex of enzymes, compared to a wild-type bacterial cell, and (b) reduced activity of pyruvate oxidase compared to a wild-type bacterial cell; and culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.
 27. The method of claim 26 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 28. The method of claim 27 wherein the additional carbon source comprises succinate.
 29. The method of claim 27 wherein the metabolite comprises pyruvate.
 30. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing a bacterial cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, (b) reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a wild-type bacterial cell, and (c) reduced activity of pyruvate oxidase compared to a wild-type bacterial cell; and culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.
 31. The method of claim 30 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 32. The method of claim 31 wherein the additional carbon source comprises succinate.
 33. The method of claim 30 wherein the metabolite comprises pyruvate.
 34. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing a bacterial cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, (b) reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a wild-type bacterial cell, (c) reduced activity of pyruvate oxidase compared to a wild-type bacterial cell, and (d) added or increased NADH oxidase activity; and culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.
 35. The method of claim 34 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 36. The method of claim 35 wherein the additional carbon source comprises succinate.
 37. The method of claim 34 wherein the metabolite comprises pyruvate.
 38. A method for making pyruvate comprising: providing a bacterial cell wherein the gene encoding at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes is knocked out; and culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.
 39. The method of claim 38 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 40. The method of claim 39 wherein the additional carbon source comprises succinate.
 41. The method of claim 38 wherein the bacterial cell further exhibits reduced activity of pyruvate oxidase.
 42. The method of claim 38 wherein the bacterial cell further exhibits reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase).
 43. The method of claim 38 wherein the bacterial cell further exhibits increased or added activity of NADH oxidase.
 44. The method of claim 38 wherein the bacterial cell further exhibits reduced activity of pyruvate oxidase and reduced activity of PEP carboxylase.
 45. The method of claim 38 further wherein the bacterial cell further exhibits reduced activity of pyruvate oxidase, reduced activity of PEP carboxylase, and increased or added activity of NADH oxidase.
 46. The method of claim 38 wherein the metabolite comprises pyruvate.
 47. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising: providing an E. coli cell wherein the gene encoding at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes is knocked out; and culturing the E. coli cell in the presence of glucose and acetate to yield the metabolite.
 48. The method of claim 47 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of E. coli.
 49. The method of claim 47 wherein the additional carbon source comprises succinate.
 50. The method of claim 47 wherein the metabolite comprises pyruvate.
 51. A method for making alanine comprising: providing a bacterial cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, and (b) added or increased alanine dehydrogenase activity compared to a wild-type bacterial cell; and culturing the bacterial cell in the presence of glucose and acetate to yield alanine.
 52. The method of claim 51 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 53. The method of claim 52 wherein the additional carbon source comprises succinate.
 54. A method for making alanine comprising: providing an E. coli cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, and (b) added or increased alanine dehydrogenase activity compared to a wild-type E. coli cell; and culturing the E. coli cell in the presence of a glucose and acetate to yield alanine.
 55. The method of claim 54 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of E. coli.
 56. The method of claim 55 wherein the additional carbon source comprises succinate.
 57. A method for making diacetyl comprising: providing a bacterial cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, and (b) added or increased acetolactate synthase activity; and culturing the bacterial cell in the presence of glucose and acetate to yield diacetyl.
 58. The method of claim 57 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.
 59. The method of claim 58 wherein the additional carbon source comprises succinate.
 60. The method of claim 57 wherein the bacterial cell further exhibits added or increased NADH oxidase activity.
 61. A method for making diacetyl comprising: providing an E. coli cell exhibiting (a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type E. coli cell, and (b) added or increased acetolactate synthase activity; and culturing the E. coli cell in the presence of glucose and acetate to yield diacetyl.
 62. The method of claim 61 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of E. coli.
 63. The method of claim 62 wherein the additional carbon source comprises succinate.
 64. The method of claim 61 wherein the E. coli cell further exhibits added NADH oxidase activity. 